The acetylcholine (ACH) receptor of electric tissue and skeletal muscle transduces the binding of ACH into a change in the permeability fo the post-synaptic membrane to cations. The receptor has the polypeptide chain composition alpha2/Beta/gamma/delta. The overall objectives of the project are to determine the three dimensional structure of the receptor in the membrane and the location within this structure of the functional sites, such as the ACH binding sites and the cation- conducting channel. We will identify the amino acid residues contributing to the ACH binding sites and to the noncompetitive inhibitor (NCI) sites. We will crosslink a Cys 192 and Cys 193, which we have shown are in the ACH binding site, to other residues in the site. We will also directly affinity label other residues in this site. We will probe the NCI sites using a rapid- mixing, rapid-photolabeling technique, which allows us to label the receptor in its transient channel-open and channel-closed states. With this techniques, we will probe the mechanism of block by local anestheties and other NCIs and will test the hypothesis that the NCI sites are within the channel. The amino acid residues that react with labels directed either to the ACH sites or to the NCI sites will be located in the sequences of the chains. We will label susceptible residues in membrane-bound receptor with membrane-impermeant reagents. We will locate these residues in the sequences of the chains in order to determine the folding pattern of the chains in the membrane. We propose to determine the three dimensional structure of the receptor by x-ray crystallography. The location of the functional sites of the receptor in a high resolution structure will provide a basis for our understanding of the function of the receptor in molecular terms.